A wide variety of chemical reactions has been proposed for immobilizing proteins on solid supports.
There are three critical requirements for covalent immobilization of proteins on solid supports.
Speed of covalent immobilization facilitates processing conditions. Density of immobilized proteins on the support determines the quantity of protein potentially available for subsequent reaction. Bound activity of the immobilized protein determines the quantity of the protein actually available for subsequent reaction.
It has been known in the art that some activated polymeric supports will bind more protein under highly ionic immobilization conditions. For example, Hannibal-Friedrich et al. report that binding of albumin and gamma-globulin to oxirane-acrylic beads are maximized in the presence of a 1.0 molar aqueous potassium phosphate solution at pH at 7.6. But the immobilization required a minimum of 5 hours. (Hannibal-Friedrich et al., Biotech. Bioengineer. 22 (1980) p. 157 et seq.)
It is also known that the covalent immobilization of many useful proteins is enhanced in the presence of sulfate ions. This effect was first noted in reference to oxirane-functional methacrylate copolymers and hydroxyethyl methacrylate polymers. (Smalla et al. Biotech. Appl. Biochem. 10 (1988) p. 21).
The art has previously reported the density of immobilized protein on the support and the amount of activity bound on the support, in order to reveal efficacy of an immobilization process.
If biologically active material were inexpensive and easy to prepare and handle, one could maximize bound biological activity by maximizing the density of the biologically active material immobilized on the support. That processing regimen could ignore economic considerations.
Economic considerations also suffer from slow immobilization processes. Protein coupling to activated supports such as oxirane (epoxide), cyanogen bromide, activated thiol, aldehyde, and hydrazide required immobilization of at least 5 and as much as 72 hours; c.f. Coleman et al., J. Chromatogr. 512 (1990) p. 360 with respect to References 19-24. For example, although described as highly universal and possible at reaction times from 0.5-40 hours, each experiment reported in U.S. Pat. No. 4,775,714 (Hermann et al.) required immobilization for at least 5 hours. Thus, even with inorganic salts like ammonium sulfate or sodium sulfate present during immobilization, the covalent coupling of protein to the support may result in acceptable protein density and bound biological activity; but the immobilization process is slow.
Others have taught the use of high concentrations of polyvalent anion in a two step reaction to immobilize proteins on a solid, nonreactive support. U.S. Pat. No. 4,839,419 (Kraemer et al.) discloses the use of sulfates, phosphates, pyrophosphates, carbonates, chromates, citrates and tartrates to adsorb the protein to a nonreactive support followed by a crosslinking step with a reagent such as glutaraldehyde to immobilize the protein adsorbed on the support. Another process employs reactive supports and dispenses with the need for the protein crosslinking step. Examples 3 and 4 report bound biological activity and activity yields of 55% and 61%, respectively, for enzyme immobilizations lasting for 72 hours and and 8 hours, respectively, using a bead support having epoxy functionality.
Azlactone-functional copolymer beads, such as those described in U.S. Pat. Nos. 4,737,560 and 4,871,824 and European Patent Publication 0 392 735 (all Heilmann et al.), have a high capacity for immobilizing protein densely while retaining significant bound biological activity. This capacity is largely independent of the degree of azlactone functionality, within the range of 1-3 meq/g of mass of beads. Further, as described in each Heilmann et al. patent or publication identified above, the attaching reaction of an azlactone-functional polymeric support with a protein in an aqueous media is very rapid, typically reaching half of the maximum amount of coupling within about five minutes after reaction initiation and completion in less than three hours.
The immobilization reactions reported in each Heilmann et al. patent or publication identified above use a low concentration (e.g., 25 mM) of phosphate buffer solution, with or without the presence in physiological concentrations of an inorganic monoanionic salt, NaCl, to carry out the immobilization. Immobilization is complete within not more than three hours.
As is conventional for protein immobilization, the immobilization in each Heilmann et al. process is followed by quenching of the remaining azlactone-functional groups on the polymeric support by reaction with an azlactone quencher, such as ethanolamine.